1. Field of the Invention
The present invention relates to a monoclonal antibody specific to tartrate-resistant acid phosphatase 5b (TRACP 5b: otherwise known as osteoclast-derived tartrate-resistant acid phosphatase), hybridomas capable of producing the monoclonal antibody, a method of detecting TRACP 5b using the monoclonal antibody, and a kit for use in the detecting method.
The monoclonal antibody of the present invention enables one to specifically assay the activity of tartrate-resistant acid phosphatase 5b and is extremely useful as a marker for bone resorption in the field of medical treatments for bone diseases and clinical diagnosis.
2. Description of the Related Arts
It is reported that tartrate-resistant acid phosphatase (TRACP: Tartrate-Resistant Acid Phosphatase EC 3.1.3.2) in serum is an acid phosphatase derived largely from osteoclasts and that the assay of TRACP activity will be useful as a measure for evaluating the function of osteoclasts. Thus, keen attention was drawn to TRACP as a marker for bone resorption (KOTSU TAISHA MARKER (Bone Metabolism Marker), edited by Masao Fukunaga, Toshitaka Nakamura & Toshio Matsumoto, 1995, published by Medical Review, Inc.) Analysis of acid phosphatases in serum on polyacrylamide gel electrophoresis has identified 6 bands, which are bands 0 to 6 from the starting point. In these 6 bands, band 5 is resistant to tartrate treatment and therefore called band 5 tartrate-resistant acid phosphatase (TRACP 5). TRACP 5 is further separated into components located at bands 5a and 5b based on electrophoretic mobility, i.e., band 5a is abundant in sialic acid-binding carbohydrate chains and band 5b having less sialic acid-binding carbohydrate chains. The TRACP 5a enzyme originating from platelets, etc. does not change its blood level, but TRACP 5b changes the blood level accompanied by bone resorption. Therefore, TRACP 5b is considered to be the only acid phosphatase derived from osteoclasts that is resistant to inhibition by tartrate (JP2002-510050A).
Abbreviation of TRACP 5b to osteoclast-derived ACP is recommended also in Clin. Chem. 47:1497, 2001. Accordingly, throughout the specification, TRACP 5b is used to mean ACP derived from osteoclasts as a marker for bone resorption and osteoclast-derived tartrate-resistant acid phosphatase and tartrate-resistant acid phosphatase 5b are collectively referred to as TRACP 5b, unless otherwise indicated.
Conventional methods of assaying TRACP activity as a biomarker of acid phosphatases to determine osteoclastic activity encounter problems in specificity, sensitivity, complicated measurements and measuring time.
In general, the assay of TRACP 5b activity can be performed by using a phosphoric acid ester as a synthetic substrate in the presence of tartrate and colorimetrically measuring the end product (alcohol or phenol) produced by the enzymatic reaction. In this assay, the tartarate inhibits prostate-derived acid phosphatase. Thus, the activity of acid phosphatase remaining is measured on the substrate and then, the TRACP activity calculated from the measurements is regarded to be the TRACP 5b activity. However, this method is not very specific, since tartrate inhibits other acid phosphatases originating from erythrocytes and platelets present in serum samples and the method includes these other acid phosphatases, in addition to the osteoclast-derived acid phosphatase. In order to improve the method above, it was proposed to pretreat serum by incubation of its 5-fold dilution at 37° C. for an hour and then measure the TRACP activity in the presence of tartrate using p-nitrophenyl phosphate (pNPP) as a substrate (NICHIDAI-ISHI, 49: 904–911, 1990; Clin. Chem., 33: 458–462, 1987). This improved method can exclude acid phosphatases derived from erythrocytes but does not exclude platelet-derived acid phosphatases. For a more specific method for assaying the activity, the present inventors previously found that there is a difference in resistance to a fluoride between TRACP 5b and erythrocyte- and platelet-derived acid phosphatases, and reported a method for determination of TRACP 5b based on this difference in resistance (JP 10-37198A). This method could eliminate any influence of erythrocyte- and platelet-derived tartrate-resistant acid phosphatases but was still affected by TRACP 5a. The method requires determination of the TRACP 5b activity by measuring the total tartrate-resistant acid phosphatase activity, and then calculating the difference between the total activity and the activity not inhibited in the presence of a fluoride. Therefore, a further improvement is desired in view of sensitivity. Another method reported involves use of an additional TRACP 5b inhibitor in the aforesaid method using a fluoride to determine the TRACP 5b activity more specifically (JP 2001-231595A). The method using a fluoride alone is more specific but a problem still remains in accuracy because the osteoclast-derived TRACP 5b activity is assayed based on the difference calculated, as in other known methods.
On the other hand, the following methods using a polyclonal antibody or a monoclonal antibody are also known for immunoassay methods to assay the TRACP 5b activity (J. Clin. Endocrinol. Metab., 71: 442–451, 1990; J. Bone Miner. Res., 13: 683–687, 1998; Immunol. Lett., 70: 143–149, 1999; J. Bone Miner. Res., 14: 464–469, 1999; Clin. Chem., 45: 2150–2157, 1999; Clin. Chem., 46: 1751–1754, 2000). In these methods, an influence of TRACP 5a is not negligible because TRACP 5a and TRACP 5b are both measured undesirably without discriminating from one another. An immunoassay for measuring TRACP 5b more specifically is reported also in WO 99/50662 and JP2002-510050A. While this method is more specific to the TRACP 5b activity, the antibody used for the immunoassay is not specific to TRACP 5b and is also reactive with TRACP 5a. The TRACP 5b activity should be assayed by calculation from the measurement data in the immunoassay, taking advantage of the difference in optimum pH between TRACP 5b and TRACP 5a. For this reason, it is a concern that inaccurate assay data may result from increased TRACP 5a levels in specimens from patients with terminal renal failure. Furthermore, the difference between normal specimens and pathologic specimens with accelerated bone resorption is so small that the sensitivity required for a bone resorption marker is not obtained (Clin. Chim. Acta, 301: 147–158, 2000).